Wu L N, Genge B R, Sauer G R, Wuthier R E
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.
Connect Tissue Res. 1996;35(1-4):309-15. doi: 10.3109/03008209609029205.
Previous studies revealed that matrix vesicles (MV) have an acid-labile nucleationally active core (ALNAC) essential for mineral formation; current studies were aimed at characterizing and reconstituting ALNAC. SDS-PAGE and FTIR analyses revealed the presence of lipids, proteins and amorphous calcium phosphate (ACP) in ALNAC. Extraction with chloroform-methanol reduced, but did not destroy MV calcification; treatment with chloroform-methanol-HCl destroyed all activity. This acidic solvent extracted the annexins, (phosphatidylserine (PS)-dependent Ca(2+)-binding proteins), and dissociated PS-Ca(2+)-Pi complexes present in the MV. Attempts to reconstitute ALNAC, centered on the Ca(2+)-PS-Pi complex. Various pure lipids, electrolytes and proteins were combined to form a synthetic nucleationally active complex (SNAC), analyzing the rate of Ca2+ uptake. Inclusion of phosphatidylethanolamine (PE) or sphingomyelin (SM) with PS, or Mg2+ or Zn2+ with Ca2+, strongly inhibited activity; incorporation of annexin V increased SNAC activity. Thus, approaching from either deconstruction or reconstruction, it appears that ALNAC is composed of ACP complexed with PS and the annexins. Other lipids, proteins and electrolytes modulate its activity. These findings also indicate how ALNAC must be formed in vivo.
先前的研究表明，基质小泡（MV）具有对矿物质形成至关重要的酸不稳定成核活性核心（ALNAC）；当前的研究旨在对ALNAC进行表征和重建。SDS-PAGE和FTIR分析显示ALNAC中存在脂质、蛋白质和无定形磷酸钙（ACP）。用氯仿-甲醇萃取可降低但不会破坏MV钙化；用氯仿-甲醇-HCl处理会破坏所有活性。这种酸性溶剂提取了膜联蛋白（依赖磷脂酰丝氨酸（PS）的Ca(2+)结合蛋白），并解离了MV中存在的PS-Ca(2+)-Pi复合物。以Ca(2+)-PS-Pi复合物为中心尝试重建ALNAC。将各种纯脂质、电解质和蛋白质组合形成合成成核活性复合物（SNAC），分析Ca2+摄取速率。将磷脂酰乙醇胺（PE）或鞘磷脂（SM）与PS一起加入，或Mg2+或Zn2+与Ca2+一起加入，会强烈抑制活性；加入膜联蛋白V可提高SNAC活性。因此，无论是从解构还是重建的角度来看，ALNAC似乎是由与PS和膜联蛋白复合的ACP组成。其他脂质、蛋白质和电解质调节其活性。这些发现也表明了ALNAC在体内必须如何形成。
